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Constitutive aryl hydrocarbon receptor signaling constrains type I interferon–mediated antiviral innate defense

Nature Immunology volume 17, pages 687–694 (2016)Cite this article

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Abstract

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic activity of many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood. Here we demonstrate that constitutive AHR signaling negatively regulates the type I interferon (IFN-I) response during infection with various types of virus. Virus-induced IFN-β production was enhanced in AHR-deficient cells and mice and resulted in restricted viral replication. We found that AHR upregulates expression of the ADP-ribosylase TIPARP, which in turn causes downregulation of the IFN-I response. Mechanistically, TIPARP interacted with the kinase TBK1 and suppressed its activity by ADP-ribosylation. Thus, this study reveals the physiological importance of endogenous activation of AHR signaling in shaping the IFN-I-mediated innate response and, further, suggests that the AHR-TIPARP axis is a potential therapeutic target for enhancing antiviral responses.

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Figure 1: Endogenously activated AHR signaling modulates IFN-I-mediated antiviral response.
Figure 2: AHR activation by endogenous ligands suppresses virus-induced IFN-I response.
Figure 3: TIPARP mediates AHR-dependent downregulation of IFN-I induction.
Figure 4: TIPARP deficiency robustly induces IFN-I response to viral infection.
Figure 5: TIPARP regulates TBK1 activity via its post- translational modification.
Figure 6: Constitutive activation of the AHR-TIPARP axis modulates interferon-mediated antiviral innate defense.

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Acknowledgements

We thank C.A. Bradfield for Ahr+/+ and Ahr−/− MEFs, A. Kimura and Y. Fujii-Kuriyama for Ahr−/− mice, J. Miyazaki for the pCAGGS vector, A. Miyawaki for the pCAGGS-YFP vector, T. Fujita for the p-125Luc vector, the Japan Red Cross society for whole blood samples, T. Kubota for pcDNA3.1(-)IRF-3/5D-FLAG, T. Miyazaki for influenza virus (strain A/Puerto Rico/8/34) and H. Kitamura, D. Kamimura and M. Murakami for technical advice on mouse experiments. We are also grateful for financial support from the Ministry of Health, Labour and Welfare of Japan (grant-in-aid to A. Takaoka); the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant-in-aid for scientific research (A) (25253030) and grant-in-aid for scientific research on innovative areas (25115502, 23112701) to A. Takaoka; grant-in-aid for young scientists (A) (25713032) to S.H.); IRYO HOJIN SHADAN JIKOKAI (granted by H. Tanaka and N. Takayanagi) to A. Takaoka; the Kato Memorial Bioscience Foundation to A. Takaoka; the Yasuda Medical Foundation to A. Takaoka; the Takeda Science Foundation to A. Takaoka; Akiyama Life Science Foundation to S.H.; Canadian Institutes of Health Research (CIHR) grant (496441) to T.H.W.; a Canadian Institutes of Health Research operating grant (MOP-125919) to J.M.; a Canadian Institutes of Health Research New Investigator Award and an Early Researcher Award from the Ontario Ministry of Innovation to J.M. and the Waksman Foundation of Japan to A. Takaoka.

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Author notes

  1. Taisho Yamada and Hiromasa Horimoto: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Signaling in Cancer and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

    Taisho Yamada, Hiromasa Horimoto, Takeshi Kameyama, Sumio Hayakawa, Hiroaki Yamato, Masayoshi Dazai & Akinori Takaoka

  2. Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

    Hiromasa Horimoto, Hiroaki Yamato & Masayoshi Dazai

  3. Molecular Medical Biochemistry Unit, Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan

    Takeshi Kameyama, Sumio Hayakawa & Akinori Takaoka

  4. Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan

    Ayato Takada

  5. Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Japan

    Ayato Takada & Hiroshi Kida

  6. Department of Disease Control, Laboratory of Microbiology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

    Hiroshi Kida

  7. Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada

    Debbie Bott, David Hutin & Jason Matthews

  8. Department of Immunology, University of Toronto, Toronto, Ontario, Canada

    Angela C Zhou & Tania H Watts

  9. Health Sciences University of Hokkaido, Toubetu, Japan

    Masahiro Asaka

  10. Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

    Jason Matthews

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Contributions

T.Y., H.H., T.K., S.H., H.Y., M.D. and M.A. carried out most of the experiments and analyzed data. A. Takada and H.K. provided VSV, NDV, SeV and advice. J.M. offered technical advice on the ADP-ribosylation assay and provided immortalized wild-type and Tiparp−/− MEFs. D.B., A.C.Z., D.H., T.H.W. and J.M. conducted FluV infection experiments with Tiparp−/− mice. A. Takaoka supervised the project, designed experiments and wrote the manuscript with critical input from all authors, and all authors contributed to discussing the results.

Corresponding author

Correspondence to Akinori Takaoka.

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Integrated supplementary information

Supplementary Figure 1 AHR-induced suppression of type I IFN response.

(a) IFN-α production by infection with the indicated viruses for 24 h in Ahr+/+ or Ahr−/− MEFs. n = 3 per group. (b) Quantitative RT-PCR analysis of Ifnb1 mRNA expression in Ahr+/+ or Ahr−/− MEFs after the indicated time periods of 3pRNA stimulation. n = 3 per group. (c) IFN-β and IFN-α production in response to the indicated viral MAMPs for 24 h in Ahr+/+ or Ahr−/− MEFs transfected with control (C), or mouse AHR expression vector (A). n = 3 per group. (d) IFN-β production by infection with the indicated viruses for 24 h in Ahr+/+ or Ahr−/− MEFs transfected with control (C), or mouse AHR expression vector (A). n = 3 per group. (e) 3pRNA-induced phosphorylation levels of TBK1 and IRF-3 in Ahr+/+ or Ahr−/− MEFs were measured by immunoblotting with anti-specific antibodies. Rescue experiment was also performed by exogenous expression of wild-type (WT) AHR in Ahr−/− MEFs. (f) IFN-β production by 3pRNA stimulation (left) or FluV infection (right) for 24 h in Ahr+/+ or Ahr−/− splenocytes. n = 3 per group. (g) Ahr+/+ or Ahr−/− BMDMs were stimulated with poly(rI:rC) without any transfection reagent for 24 h, and IFN-β production was measured by ELISA. In this case, poly(rI:rC) was used as a ligand for TLR3. n = 3 per group. (h) The expression levels of indicated mRNA were measured by qRT-PCR in Ahr+/+ or Ahr−/− MEFs (left), or DMSO or CH-223191-treated WT MEFs (right). n = 3 per group. (i) IFN-β production by FluV infection (left) or 3pRNA stimulation (right) for 24 h in WT BMDMs pretreated with DMSO or CH-223191. n = 3 per group. (j) Induction of Ifnb1 mRNA by VSV infection or 3pRNA stimulation for 6 h was evaluated by qRT-PCR in WT MEFs pretreated with DMSO or CH-223191. n = 3 per group. *P < 0.05, **P < 0.01 as compared with control (Student’s t-test). Data are representative of at least two independent experiments with similar results (mean ± s.d.). ND, not detected.

Supplementary Figure 2 Endogenous AHR-ligand-induced suppression of type I IFN response.

(a) Induction of Ifna1 mRNA by 3pRNA stimulation was evaluated by qRT-PCR in MEFs pretreated with Control (C), Kyn (50, 100, and 200 μM) or FICZ (0.1, 1, and 25 nM). n = 3 per group. (b) Ratio of the media concentrations of Kyn and Tryptophan (Trp) in MEFs at 24 h after treatment with a TDO inhibitor 680C91 (10 μM). n = 3 per group. (c) Ratio of the media concentrations of Kyn and Trp in BMDMs at 24 h after treatment with an IDO inhibitor 1-MT (left). ELISA of IFN-β response by FluV infection in Ahr+/+ or Ahr−/− BMDMs pretreated with DMSO or 1-MT (right). n = 3 per group. (d) Gene expression of the indicated AHR-inducible genes was analyzed by qRT-PCR in MEFs infected with VSV for 6 h. Oas1a and Rsad2 mRNA expression levels were measured as positive controls. n = 3 per group. (e) Quantitative RT-PCR analysis of 3pRNA-induced Ifnb1 mRNA expression in MEFs pretreated with Kyn for 2 h in the absence (DMSO; left) or presence of cycloheximide (CHX; right). Data are shown as the percentage of control. n = 3 per group. (f) Knockdown efficiency of the indicated siRNAs was evaluated by qRT-PCR analysis in MEFs. n = 3 per group. (g) Quantitative RT-PCR analysis of TIPARP mRNA expression levels in human primary CD14+ monocytes treated with FICZ for 2 h. n = 3 per group. (h) MEFs were treated with Kyn (left) or FICZ (right). Gene expression of the indicated PARP-superfamily members was analyzed by qRT-PCR. Zc3hav1 has two isoforms (Zap and Zaps). n = 3 per group. (i) Quantitative RT-PCR analysis of Tiparp mRNA expression levels in the organs of mice at 12 h after intraperitoneally injection with FICZ. Mean values are represented by horizontal bars. n = 4 per group. *P < 0.05, **P < 0.01 as compared with control (Student’s t-test). Data are representative of at least two independent experiments with similar results (mean ± s.d.). ND, not detected. NS, not significant.

Supplementary Figure 3 TIPARP-mediated regulation of type I IFN response to stimulation with viral mimetic nucleic acids.

(a) HEK293T cells expressing control plasmid (Control) or TIPARP were treated with 3pRNA for the indicated time periods, and then assayed for IFNB1 and IFNA1 mRNA expression by qRT-PCR. n = 3 per group. (b) Luciferase activity of a p-125Luc reporter plasmid after 24 h of 3pRNA stimulation in HEK293T cells transfected with control plasmid (C) or the increasing doses of TIPARP expression vector (0.01, 0.1, and 1 μg). n = 3 per group. (c) Quantitative RT-PCR analysis of IL8 mRNA expression levels after 24 h of IL-1β stimulation in HEK293T cells transfected with control (C) or the increasing doses (0.01, 0.1, and 1 μg) of TIPARP expression vector. n = 3 per group. (d) Knockdown efficiency of siTIPARP-1 was evaluated by qRT-PCR analysis of TIPARP mRNA expression levels in HEK293T cells (left). Quantitative RT-PCR analysis of IFNB1 mRNA induction by 3pRNA stimulation in HEK293T cells treated with siControl or siTIPARP-1 (right). n = 3 per group. (e) Quantitative RT-PCR analysis of Ifna1 mRNA induction in response to the indicated viral MAMPs in Tiparp+/+ or Tiparp−/− MEFs. n = 3 per group. (f) Tiparp+/+ or Tiparp−/− BMDMs were stimulated with poly(rI:rC) without any transfection reagent for 24 h and IFN-β production was measured by ELISA. In this case, poly(rI:rC) was used as a ligand for TLR3. n = 3 per group. **P < 0.01 as compared with control (Student’s t-test). Data are representative of at least two independent experiments with similar results (mean ± s.d.). ND, not detected. NS, not significant.

Supplementary Figure 4 Colocalization between TIPARP and TBK1 in the perinuclear region.

(a) Interaction of recombinant TBK1 protein (rTBK1) with recombinant GST or GST-TIPARP was analyzed by GST-pull down and immunoblotting with anti-TBK1 or anti-GST antibodies. Inputs were also immunoblotted by anti-TBK1. (b) Intermolecular FRET analysis for the interaction between TIPARP and TBK1. HeLa cells transiently expressing mCherry-tagged TBK1 together with YFP alone (top row) or YFP-tagged TIPARP (bottom row) were analyzed by fluorescence microscopy. Representative fluorescence images of YFP, mCherry and FRETc (corrected FRET; displayed in pseudo-color mode) are shown from left to right. FRETc/YFP values were calculated and plotted as an aligned dot plot. Mean values are represented by horizontal bars. n = 15 per group. **P < 0.01 as compared with control (Student’s t-test). (c) HEK293T cells transfected with HA-tagged TIPARP were pretreated with Leptomycin B (200 μg/ml) for 2 h, and subjected to 3pRNA stimulation for 4 h. The nuclear/cytosol distribution of TIPARP was assessed by immunoblot analysis. The relative band intensities of the cytosolic TIPARP quantified by a densitometer were normalized to those of cytosolic α-tubulin, and depicted in graphs. (d) HEK293T cells cotransfected with the expression vectors for Flag-tagged TBK1 (WT) or its deletion mutants (KD or ULD+CC) (illustrated in upper), together with HA-tagged TIPARP expression vector, were subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-HA or anti-Flag. Whole cell lysates (WCL) were also immunoblotted by anti-HA. KD, kinase domain; ULD, ubiquitin-like domain; CC, coiled-coil. (e) Interaction of endogenous TBK1 with HA-tagged TIPARP (WT) or its deletion mutants (N-TIPARP or C-TIPARP) (illustrated in upper) in HEK293T cells was analyzed by immunoprecipitation with anti-HA antibody and immunoblotting with anti-TBK1 or anti-HA. Whole cell lysates (WCL) were also immunoblotted by anti-TBK1. (f) 3pRNA-induced phosphorylation levels of TBK1 and IRF-3 in Tiparp+/+ or Tiparp−/− MEFs were measured by immunoblotting with anti-specific antibodies. Data are representative of at least two independent experiments with similar results.

Supplementary Figure 5 The TBK1 kinase domain but not the ULD+CC domain is ADP-ribosylated in the presence of TIPARP protein.

(a,b) Tiparp+/+ or Tiparp−/− MEFs pretreated with Biotin-NAD were stimulated with 3pRNA for the indicated time periods. Pull-down assay was performed, followed by immunoblotting with anti-TBK1 antibody. Whole cell lysates (WCL). (c) ADP-ribosyltransferase activity of TIPARP protein was examined by incubating recombinant GST-TIPARP protein with recombinant TBK1 protein (WT) or its deletion mutants (KD or ULD+CC) in the presence of 32P-NAD+. These mixtures were subjected to SDS-PAGE and autoradiography. β-NAD+ was added as a cold competitor. KD, kinase domain; ULD, ubiquitin-like domain; CC, coiled-coil. ns, non-specific bands. (d,e) Tiparp−/− MEFs rescued by reintroduction with TIPARP (WT) or its mutant (H532A) expression vector were stimulated with the indicated viral MAMPs for 24 h, and the production of IFN-β (d) and IFN-α (e) was evaluated by ELISA. n = 3 per group. (f) WT, Ahr−/− or Tiparp−/− MEFs pretreated with DMSO or 680C91 for 24 h were uninfected or infected with VSV for 6 h. The expression levels of Ifnb1 mRNA were measured by qRT-PCR. Data are shown as the percentage of control (DMSO). n = 3 per group. (g) Quantitative RT-PCR analysis of Tiparp mRNA expression levels in lungs of mice at 4 h after intraperitoneally injection with corn oil (Control) or CH-223191 (100 μg per mouse), related to Fig. 6d,e. n = 3 per group. **P < 0.01 as compared with control (Student’s t-test). Data are representative of at least two independent experiments with similar results (mean ± s.d.). ND, not detected. NS, not significant.

Supplementary Figure 6 Schematic representation of constitutive AHR signaling for the TIPARP-mediated modulation of antiviral IFN response.

AHR signaling activated by endogenous ligands (Kyn, etc.) constitutively upregulates the levels of TIPARP, which interacts with TBK1 for the downregulation of TBK1 activity by ADP-ribosylation. This AHR-TIPARP axis plays a key role for tuning nucleic acid sensor-mediated type I IFN induction. TDO, tryptophan-2,3-dioxygenase; IDO, indoleamine-2,3-dioxygenase; Kyn, kynurenine; FICZ, 6-formylindolo[3,2-b]carbazole; AHR, aryl hydrocarbon receptor; TIPARP, TCDD-inducible poly(ADP-ribose) polymerase; TBK1, TANK-binding kinase 1; KD, kinase domain; ULD, ubiquitin-like domain; CC, coiled-coil; IRF-3, interferon regulatory factor-3; IFN, interferon.

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Yamada, T., Horimoto, H., Kameyama, T. et al. Constitutive aryl hydrocarbon receptor signaling constrains type I interferon–mediated antiviral innate defense. Nat Immunol 17, 687–694 (2016). https://doi.org/10.1038/ni.3422

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